This application claims priority under 35 USC §119(e) and 37 C.F.R. §1.78 to pending provisional patent application No. 60/259,211, filed 01 Jun. 2001.
In general, the present invention relates to techniques for monitoring halogenated organic chemicals (pollutants, pesticides, etc.) in soil, as well as groundwater, waste waters, and other aqueous environments. More particularly, the invention is directed to an improved distal tip having a transducer to which a biocomponent comprising a dehalogenase (selected for dehalogenation of a selected analyte of interest) is immobilized, treated, and/or stabilized for monitoring continuously in situ soil or an aqueous environment to detect the presence and/or concentration of the analyte, such as any of the s-triazine pesticides, including the chlorinated herbicide atrazine (used to control broadleaf and grassy weeds), simazine, terbuthylazine, propazine, cyanazine, deethylatrazine, and deisopropylatrazine, plus other s-triazines (including those in TABLE 1), lindane, and DDT. Disclosed is a novel technique of measuring an analyte and associated biosensor capable of measuring pH (hydrogen ion) and halide ion concentration in soil and aqueous environments to detect the concentration, or collect other information about, the analyte. In one aspect of the invention, focus is on a unique biosensor including a fiber optic element (an optical fiber or bundle), the tip of which has a layer of a bacteria atop a layer of a pH-sensitive fluorophore (dye). The bacteria is selected such that it carries an enzyme to catalyze a reaction with the halogenated compound of the analyte, releasing either protons (and causing a detectable pH change) or a measurable halide ion concentration. Further, prior to being ‘glued’ (immobilized or otherwise affixed) to the tip of the fiber optic transducer, the bacteria layer is specially treated and/or stabilized.
Currently available techniques to measure analytes, and more-particularly pollutants, in groundwater include ex situ laboratory measurements which generally have a long response time and are expensive, or use of immunoassay kits which can be quite inaccurate and also expensive. In Campbell, 1998 entitled “The Development of Biosensors for the Detection of Halogenated Groundwater Contaminants.” Spring 1998, submitted by D. W. Campbell in fulfillment of the requirements for the Degree of Master of Science at Colorado State University, available from Morgan Library at the Colorado State University in Fort Collins, Colo., reference is made to a pH optode structure featuring the reaction illustrated schematically in Campbell, 1998 (labeled FIG. 2.4): the cleavage of halide ion X− and proton H+ from a halogenated hydrocarbon by the appropriate hydrolytic dehalogenase. An earlier reference entitled “Multicomponent fiberoptical biosensor for use in hemodialysis monitoring” (Cord Muller, et al.) employed a pH optode-type biosensor structure limited to the use of urease as a catalyst (urea is split into ammonia & CO2): the bifunctional reagent glutaraldehyde was used to bind the urease directly to the head of a pH optode.
Chemical biosensors are miniaturized analytical devices, which can deliver real-time and on-line information on the presence of specific compounds or ions in complex samples. Usually an analyte recognition process takes place followed by the conversion of chemical information into an electrical or optical signal. Two popular classes of chemical sensors used today are electrochemical transduction type. (amperometric, potentiometric, including ion-selective electrodes (ISE), field effect transistors (FETs), gas-sensing electrodes, etc., and conductimetric) and optical transduction type (including pH optodes). They are used during laboratory analysis as well as in industry, process control, physiological measurements, and environmental monitoring. The basic principles of operation of the chemical sensors utilizing electrochemical and optical transduction are quite well understood. In developing biosensors for general manufacture and commercial use, longevity and stabilization of the biocomponent are critical. It is preferable to have a stable, long-lived biosensor that can stand prolonged storage as well as perform well in use for a selected period of time. Among the biocomponent possibilities, enzymes, though very selective, fall on the lower end of the ‘stability spectrum’.
The s-triazine compounds include many pesticides. Within the s-triazine family (which includes both pesticides and non-pesticide groups, see TABLE 1), atrazine is most widely used, although others include simazine, terbuthylazine, propazine, cyanazine, deethylatrazine, and deisopropylatrazine as well as others in FIG. 1, a depiction of several pathways derived from general knowledge of atrazine degradation. S-triazines are characterized by a symmetrical hexameric ring consisting of alternating carbon and nitrogen atoms.
TABLE 1Non-pesticide s-triazine groups with comments about use and biodegradability.Cyanuric (isocyanuric) acids: N-Chlorination of Cyanuric acid at the R1, R2,and R3 sites yields chloroisocyanurates that are used as disinfectants (inswimming pools and hot-tubs), sanitizers (in household cleansers and automaticdishwashing compounds), and bleaches (in both the industrial and householdbleaching of fabrics). The most common chloroisocyanurates are Trichloro andDichloro isocyanuric acid(TCCA, DCCA) and Sodium dichloroisocyanuric acid(SDCC). Triallyl isocyanurate (R1, R2, and R3 = propenyl) is used as a crosslinking agentfor poly(vinyl chloride) and other systems.Methylamine (also on the metapathway map) and N-substituted methylamines aresometimes used as finishing agents for textiles. Nitramine explosives: Cyclotrimethylenetrinitramine (RDX) is an explosive and apropellant used in military rockets. The partial biodegradation of RDX by mixedmicrobial culture is reported in (Binks et al 1995). Triazone: A cyclic urea used as a cross linking agent in textile finishing. 1,3-di-methylol-5-alkyltriazone is still widely used for this purpose. Cross linkingagents are used in the preparation of textiles to induce “memory” and to addluster.